Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found in the text and at the end of this application, preceding the sequence listing and the claims.
The following standard abbreviations are used throughout to refer to amino acids:
CD40 (1) is a receptor on B cells that interacts with the helper T cell surface protein CD40L (CD40 ligand, also known as T-BAM, gp39, or TRAP) (2-4). CD40L is found particularly on lymphoid follicle CD4+ T lymphocytes, where it delivers a contact-dependent signal that stimulates B cell survival, growth, and differentiation (2-4). Signaling through CD40 rescues B cells from apoptosis induced by Fas (CD95) or by cross-linking of the immunoglobulin M (IgM) complex (5); it also induces B cells to differentiate and to undergo Ig isotype switching (3) and to express CD80 (B7 or BB-1) (6). The crucial role of CD40L-CD40 interaction is illustrated by humans with defects in CD40L, who manifest a serious immune deficiency syndrome, the X-linked hyper-IgM syndrome (HIGMX-1) characterized by an absence of IgG, IgA, and IgE, elevated IgM, and no lymphoid follicles (7). The essential roles of CD40L and CD40 in the phenotype of HIGMX-1 syndrome has been confirmed by targeted disruption of either CD40L (8) or CD40 (9) in mice. In addition to B cells, CD40 is also expressed by follicular dendritic cells (10), dendritic cells (11), activated macrophages (12), epithelial cells (including thymic epithelium) (13), and a variety of tumor cells.
Stimulation of CD40 causes the tyrosine phosphorylation of multiple substrates including Src family kinases such as p53-p56lyn, activates multiple serine-threonine-specific protein kinases, and induces the phosphorylation of phospholipase C-xcex32 and of phosphoinositide-3xe2x80x2 kinase (14).
In mice the CD40 cytoplasmic tail is necessary for signaling (15). Proteins which interact with the cytoplasmic tail of CD40 have been described (H. M. Hu, et al., J. Biol. Chem. 269: 30069 (1994); and G. Mosialos, et al., Cell 80:389 (1995)). These proteins are the same as CRAF1.
This invention provides a protein comprising CRAF1 truncated by from about 323 to about 414 amino acid residues at the amino terminus, or a variant thereof capable of inhibiting CD40-mediated cell activation.
This invention provides a method of inhibiting activation by CD40 ligand of cells bearing CD40 on the cell surface, comprising providing the cells with an agent capable of inhibiting CD40-mediated intracellular signaling, the agent being present in an amount effective to inhibit activation of the cells.
This invention provides a method of providing a subject with an amount of a protein comprising CRAF1 truncated by from about 323 to about 414 amino acid residues at the amino terminus, or a variant thereof effective to inhibit activation by CD40 ligand of cells bearing CD40 on the cell surface in the subject, comprising: introducing into CD40-bearing cells of the subject, a nucleic acid sequence encoding the protein under conditions such that the cells express in the subject an activation inhibiting effective amount of the protein.
This invention provides a method of treating a condition characterized by an aberrant or unwanted level of CD40-mediated intracellular signaling, in a subject, comprising providing the subject with a therapeutically effective amount of an agent capable of inhibiting CD40-mediated intracellular signaling in cells bearing CD40 on the cell surface.
This invention provides a nucleic acid molecule encoding a protein comprising CRAF1 truncated by from about 323 to about 414 amino acid residues at the amino terminus, or a variant thereof capable of inhibiting CD40-mediated cell activation.
This invention provides a method of identifying an agent capable of inhibiting CD40-mediated intracellular signaling in a cell expressing CD40 on the cell surface, comprising providing the cell with the agent under conditions permitting activation of the cell in the absence of the agent, and determining decreased or absent activation, thereby identifying an agent capable of inhibiting CD40-mediated intracellular signaling in a cell expressing CD40 on the cell surface.